The chemokine CXCL12 (SDF-1) and its cognate receptor CXCR4 are involved in a large number of physiological processes including HIV infectivity, inflammation, tumorigenesis, stem cell migration, and autoimmune diseases. There has been one approved drug that targets CXCR4, an antagonist which is used for stem cell mobilization prior to autologous stem cell transplantation. However, this drug has the potential for several important side effects such as leukocytosis and possible effects on the cardiovascular system. New CXCR4-acting agents could provide therapeutics against a wide variety of diseases.
Although inflammatory cytokines were originally named for their important role in the regulation of immune cell function, it is now clear that they also have important effects in many other tissues including the nervous system. The “chemotactic cytokines,” or chemokines are a case in point. These small secreted proteins exert their effects through the activation of a family of Gprotein coupled receptors (GPCRs) and were originally shown to be key mediators of the inflammatory response due to their powerful chemoattractant effects on different classes of leukocytes. However, it is now known that the most ancient function of chemokine signaling concerned their ability to regulate the migration and development of stem cells. Indeed, CXCR4 chemokine receptor signaling is important in the development of all tissues.(ref. 1; herein incorporated by reference in its entirety) For example, it has been demonstrated that SDF-1/CXCR4 is important for the formation of the hippocampal dentate gyrus (DG)1a, and the importance of CXCR4 signaling in the development of many structures in both the central and peripheral nervous systems has also been demonstrated.(ref. 1; herein incorporated by reference in its entirety) Moreover, the developmental functions of CXCR4 signaling are still apparent in the adult.(refs. 1 b, 1c; herein incorporated by reference in their entireties) The role of CXCR4 in anchoring haemopoietic stem cells in the bone marrow is a well-known example of this. In addition, it is also clear that CXCR4 plays an important role in the regulation of cancer metastasis.(ref. 1; herein incorporated by reference in its entirety) Of great significance is that the CXCR4 receptor acts as a receptor for HIV-1 allowing it to infect lymphocytes and other cells.
Inhibition of CXCR4 signaling may be an important therapeutic strategy in many circumstances including cancer, HIV-1 pathogenesis, and several functions within the nervous system.(refs.1a, 1b; herein incorporated by reference in their entireties) A large number of investigations have sought to produce CXCR4 antagonists for therapeutic purposes.(ref. 2; herein incorporated by reference in its entirety) In addition, CXCR4 agonists or partial agonists which can rapidly desensitize CXCR4 receptors might also inhibit CXCR4 signaling by such a mechanism and may also have other important signaling consequences. However, apart from peptide mimics, no small molecule CXCR4 agonists have been reported.
Previous approaches to the discovery of new CXCR4 antagonists have relied largely on ligand-based techniques because GPCRs are notoriously difficult to crystallize.;ref. 3; herein incorporated by reference in its entirety) CXCR4 antagonists have been discovered through modification of AMD3100,(ref. 2c; herein incorporated by reference in its entirety) peptide deconstruction,(ref. 2d; herein incorporated by reference in its entirety) or high-throughput screening (HTS).(refs.2e, 2f; herein incorporated by reference in their entireties) Recently, several crystal structures of CXCR4 were solved that provide valuable insight into its ligand binding.(ref. 4; herein incorporated by reference in its entirety) Analysis of the binding mode confirmed the importance of the charged residues identified from mutation studies (ref. 5; herein incorporated by reference in its entirety) and in addition, characterized a number of important hydrophobic interactions. Comparing virtual high-throughput screening (vHTS) using a protein homology model and the actual crystal structure indicates that the crystal structure provided a significantly better receptor for docking than did the model.(ref. 6; herein incorporated by reference in its entirety).